Preparation and storage of pancreatic islets

ABSTRACT

A method, a solution and a chamber for the preparation and storage of pancreatic islets. The method includes contacting a pancreas with a warm collagenase solution, digesting the pancreas in the warm collagenase solution to form warm digest, adding cold preservative solution to the warm digest, agitating the warm digest/cold preservative solution at a temperature between about 0° and 15° C., to thereby further digest the partially digested pancreas included in the warm digest, to form cold digest and collecting liquid from the cold digest to form isolated islets. The cold preservative solution and a pancreatic islet preservative solution of the present invention include D-mannitol, K-lactobionate and a buffer.

This invention was made with government support under grant DK20827,awarded by The National Institutes of Health. The United Statesgovernment has certain rights in this invention.

This is a division of application Ser. No. 08/420,005, filed Apr. 10,1995 now U.S. Pat. No. 5,679,565.

FIELD OF THE INVENTION

This invention is directed at the preparation and storage of pancreaticislets for transplantation into diabetic patients.

BACKGROUND OF THE INVENTION

Pancreatic islet transplantation has the potential to be the mostphysiologically advantageous and minimally invasive procedure fortreatment of type I diabetes mellitus. However, despite progressivelyincreasing numbers of islet transplants in these patients, the endocrinefunction established by the transplant is far from optimum. In order forthis approach to be a clinically acceptable diabetes therapy, severaltechnical and immunological problems need to be solved.

Donor islet preparation is the first critical step to provide asufficient number of high quality islets for transplantation.Large-scale islet preparation from the pancreas of large animal species,including dogs, pigs and humans, has become possible through thedevelopment of highly automated procedures. Islet isolation, involvingthe digestion of pancreatic tissue and the purification of islets, is,in particular, the most important process that influences the outcome oftransplants.

Pancreatic islets are usually transplanted into diabetic patients whoalso need a kidney or other solid organ transplant who are alreadybeing, or will be treated with immunosuppressants in order to preventgraft rejection. Despite many attempts, to date only a small fraction ofislet allografts have functioned for a prolonged period. One of themajor reasons for this failure appears to be an insufficient number ofislets used for transplantation. The current recommendation by theInternational Islet Transplant Registry is to transplant more than 6,000islets, equivalent to 150 μm in size, per kg of the recipient's bodyweight in order to achieve long-term maintenance of euglycemia. Tofulfill this requirement, islets from multiple donors have often beenused. However, a more desirable approach would be to derive both kidney(or other organs) and islets from the same donor in order to avoid anadditional antigenic load and therefore, decrease the possibility ofrejection. This would require the isolation of a large number of highquality islets from a single human pancreas.

Large-scale islet isolation from the human pancreas has become possiblewith advances in technology and the availability of high qualitycollagenase used in their preparation. However, even with theseimprovements, the islet yield from a single pancreas is ofteninsufficient for transplantation. It is desirable to develop a methodfor isolation of pancreatic islets and storage of the islets so thattransplants can be prepared from a single donor.

SUMMARY OF THE INVENTION

The present invention is directed at a method, a solution and a chamberfor the preparation and storing of pancreatic islets.

The method of the present invention comprises contacting a pancreas witha warm collagenase solution, digesting the pancreas in the warmcollagenase solution to form warm digest, adding cold preservativesolution to the warm digest, agitating the warm digest/cold preservativesolution at a temperature between about 0° and 15° C., to therebyfurther digest the partially digested pancreas included in the warmdigest, to form cold digest and collecting liquid from the cold digestto form isolated islets.

The solution of the present invention comprises a sugar derivative suchas D-mannitol, K-lactobionate and a buffer.

The chamber of the present invention comprises a lower chamber for thedigestion of pancreatic pieces and a lid inserted into the lowerchamber. The lid comprises a cap, a filter attached to the cap, whereinthe filter is placed within the lower chamber when the lid is insertedinto the lower chamber, a port attached to the cap wherein digestedpancreatic pieces are decanted from the lower chamber, filtered throughthe filter and removed from the chamber through the port and a coverattached to the port to prevent contamination of the contents of thechamber.

BRIEF DESCRIPTION OF THE DRAWINGS

Features, aspects and advantages of the invention will be more fullyunderstood when considered with respect to the following detaileddescription, appended claims and accompanying drawing where:

FIG. 1 is an exploded perspective view of a digestion chamber of thepresent invention.

DETAILED DESCRIPTION

The present invention is directed at a new isolation technique whichuses an intermittent two-step digestion procedure for pancreatic tissueand includes warm and cold digestion steps. The invention is alsodirected at a chamber for use in the digestion procedure and a coldstorage solution for storage of isolated islets and digested pancreatictissues. The present invention has lead to the consistent isolation of asignificantly higher number of islets than were achieved by previouslyused methods.

For the isolation of pancreatic islets in accordance with the presentinvention tissue is digested by a two-step procedure, first by warmdigestion, then by cold digestion, as described below.

The pancreas is transported, under refrigeration or on ice, in asolution such as 30 mM raffinose, 100 mM K-lactobionate, 15 mM KH₂ PO₄,5 mM MgSO₄, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 8 mMdexamthazone and 5% (w/v) hydroxyethyl starch (HES), pH 7.4 or othersuitable transportation medium. The organ is preferably trimmed ofsurrounding membranes, vessels, fat and lymph nodes to reduce the amountof "contaminating" tissue and material from the pancreas, cannulatedwith an angiocatheter and weighed. In a preferred embodiment of thepresent invention, the organ is expanded by injecting 150-250 ml of warmcollagenase solution using a 30 ml syringe and sectioned intoapproximately 8-10 pieces. In another embodiment of the presentinvention the pancreas is soaked in or otherwise contacted with thecollagenase solution. It will also be realized by those skilled in theart that the pancreas could be processed whole rather than being cutinto pieces. In a preferred embodiment the collagenase solution includesa collagenase such as Collagenase P (Boehringer Mannheim Colo.,Indianapolis, Ind.), about 2% (v/v) heat-inactivated newborn bovineserum (NBS, Sigma Chemical Co., St Louis, Mo.), about 1 mg/ml calciumchloride and about 40 mg/dl DNase in a solution such as 5.6 mM glucose,15 mM KH₂ PO₄, 0.33 mM Na₂ HPO₄, 0.82 mM MgSO₄, 5.4 mM KCl, 137 mM NaCland 1.3 mM CaCl₂, pH 7.2, although one skilled in the art willappreciate that other collagenases and components of the collagenasesolution could be substituted or used and still successfully digest thepancreas. The concentration of collagenase is optimized with respect tothe time required to achieve digestion of the pancreas for each lotcollagenase and usually ranges from 1.5 to 2.5 mg/ml.

In a preferred embodiment, pieces of pancreas are placed in a container,such as that described in FIG. 1, containing an agitator such as glassmarbles. The chamber is gently agitated in an incubator or a water bathat about 37° C. using a shaker such as a Wrist Action Shaker Model 175provided by Burrell Co. of Pittsburgh, Pa., for about 15 minutes, untilthe pancreas is partially digested (Step 1: warm digestion phase). Whileit is preferred that the digestion be performed at 37° C. othertemperatures, at which the collagenase retains its activity, but do notresult in significant damage to the pancreatic islets, could be used,generally temperatures within the range of 35° to 38° C. are suitablefor pancreatic digestion.

Preferably the partially digested tissues/collagenase solution, isfiltered through a sheet of large-mesh (about 5 to about 10 mm) screeninto a second container and placed on ice. While filtration is preferredit will be understood that other methods of separation such as gravitysedimentation or decanting would also be applicable or, alternatively,the partially digested tissues/collagenase solution could be furtherprocessed without filtration or separation of the partially digestedtissues/collagenase solution. Cold (about 4° C.) preservation solutionis added to the second container at a volume approximately 1/3 of thedecanted volume. The preservation solution of the present inventioncomprises K-lactobionate, a sugar derivative such as D-mannitol and abuffer such as KH₂ PO₄. The preservation solution may also include amembrane stabilizer such as MgSO₄, a radical scavenger such assuperoxide dismutase, catalase, and vitamins such as vitamin C, vitaminE and nicotinamide and combinations thereof. The concentrations of thecomponents of the preservation solution are preferably in the range of25 to 50 mM for D-mannitol, 80 to 120 mM for K-lactobionate, 15 mM forKH₂ PO₄ and when used, 1 to 5 mM MgSO₄, superoxide dismutase at aconcentration where free radicals are eliminated from the solution butnot at a concentration which is so high as to be wasteful of thematerial and 1 to 10 mM for nicotinamide (LAP-1). In a preferredembodiment of the present invention the preservative solution comprises30 mM D-mannitol, 100 mM K-lactobionate, 15 mM KH₂ PO₄ and when used, 5mM MgSO₄, 30,000 units/l superoxide dismutase and 5 mM nicotinamide(LAP-1).

When the method employed uses a separation step as described above, warmcollagenase solution is again added to cover the remaining undigestedtissue and warm digestion is repeated. After about 5 minutes, allcollagenase/digested tissues in the first container are transferred intothe second container. The second container is gently shaken, for exampleby hand, at about 4° C. or on ice. About every 5 minutes, 1/3 of thesupernatant containing digested tissues is decanted, from the secondcontainer, into a collection bottle containing a cold preservativesolution as described above and preferably LAP-1 solution supplementedwith about 20% (v/v) serum such as newborn bovine serum. A preservativesolution is added to the second container at about a volume equal tothat decanted and the container is shaken again. This "cold digestion"process is repeated until all islets are freed (Step 2: cold digestionphase). If necessary, warm digestion is repeated a third time, so thatonly undigested, larger ductal structures remained.

To avoid harmful effects on digested tissues, collagenase and otherenzymes released from pancreatic acinar cells are inactivated as soon aspossible after the cold digestion step. For this purpose, digestedpancreatic tissues containing free islets are collected from the secondcontainer at 5-10 minute intervals and stored on ice in a large amountof cold preservation solution which includes a high concentration ofserum, although other methods of inactivating the enzymes are known tothose skilled in the art. Pancreatic digests are stored in apreservation solution as described above and preferably cold LAP-1solution supplemented with about 20% (v/v) NBS or other suitable serum.

In a preferred embodiment of the present invention, pancreatic digestsare suspended in a solution such as Euro-Ficoll solution such as thatsupplied by Pharmacia with a density of 1.100 g/cm³ and islets areseparated from acinar cells by discontinuous gradient centrifugation onthree layers of Euro-Ficoll solutions (densities of: 1.100, 1.087 and1.056) using a COBE2991 cell processor such as that supplied by CobeLaboratories, Inc. of Lakewood, Colo. After about 10 minutescentrifugation, islet fractions are identified and collected. Islets arewashed twice with a cold preservative solution such as LAP-1 and oncewith a medium such as RPMI1640 medium, supplied by GIBCO, containing 10%(v/v) fetal bovine serum (FBS) at room temperature. Other suitablemethods of cell purification are known to those skilled in the art andwould also be suitable for use in the present invention. The practice ofthe present invention typically provides islets with a purity of atleast 70%, islets retain at least 90% of their viability and retaintheir ability to respond to glucose stimulus.

In a second aspect of the present invention a solution for the storageand preservation of pancreatic islets has been developed. The solutionhas been designated LAP-1 and comprises K-lactobionate, a sugarderivative such as D-mannitol and a buffer such as KH₂ PO₄. Thepreservation solution may also include a membrane stabilizer such asMgSO₄, a radical scavenger such as superoxide dismutase, catalase,vitamin C or vitamin E and nicotinamide. The concentrations of thecomponents of the preservation solution are preferably in the range of25 to 50 mM for D-mannitol, 80 to 120 mM for K-lactobionate, 15 mM forKH₂ PO₄ and when used, 1 to 5 mM MgSO₄, superoxide dismutase at aconcentration where free radicals are eliminated from the solution butnot at a concentration which is so high as to be wasteful of thematerial and 1 to 10 mM for nicotinamide. In a preferred embodiment ofthe present invention the preservative solution comprises 30 mMD-mannitol, 100 mM K-lactobionate, 15 mM KH₂ PO₄ and when used, 5 mMMgSO₄, 30,000 units/l superoxide dismutase and 5 mM nicotinamide.Additionally, the solution may include trypsin inhibitors and othercomponents as desired. The pH of the solution is adjusted to about 7.3at room temperature. Newborn calf serum, or other suitable serum, isadded to a concentration of about 5 to 20% (v/v) and preferably 20%(v/v) to store digested tissues. In a preferred embodiment LAP-1comprises 30 mM D-mannitol, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mMMgSO₄, 30,000 units/l superoxide dismutase and 5 mM nicotinamide.

The present invention is also directed at a chamber for performing warmdigestion of pancreas (see FIG. 1). The chamber 10 is manufactured froma transparent material such as plastic or glass. It is preferable thatthe material is sterilizable by autoclaving or other suitablesterilization procedures well known to those skilled in the art. Thechamber 10 comprises a lower chamber 12, which in a preferred embodimentof the present invention, is a cylinder which is closed at a first end14 and open at a second end 16. In use pancreas pieces are added to thelower chamber along with collagenase solution described above.

In one embodiment of the present invention a lid 18 fits into and sealsthe second end of the lower chamber. The lid may also attach to thelower chamber by a screw fit, well known to those skilled in the art.Lid 18 comprises a cap 20 to seal the lower chamber and to prevent thecontents of the lower chamber from being contaminated. In a preferredembodiment the cap is dimensioned to fit within a channel 15 positionedaround the interior circumference of the lower chamber, adjacent to thesecond end. Cap 20, when inserted into the lower chamber fits into thechannel and is held in place at the second end of the lower chamber.

Attached to the underside, the side which is inserted into the lowerchamber, is a filter 22. The filter is attached to and spaced from thecap by posts 24. The filter is dimensioned to fit closely to theinterior wall of the lower chamber, adjacent to channel 15, when the lidis placed on the lower chamber.

The filter includes perforations 26 to allow digested material to passfrom the lower chamber, through the filter and out port 28. In apreferred embodiment of the present invention the perforations are about0.8 mm in diameter and allow smaller digested material to be decantedfrom the lower chamber. Port 28 is provided with a cover 30 which isdimensioned to slidably mate with the exterior of port 28 to seal theport to prevent contamination of the interior of the chamber. The covermay also attach to the port by a screw fit, well known to those skilledin the art.

EXAMPLE 1 Isolation of Pancreatic Islets and Comparison of StorageSolutions

Ten consecutively harvested human pancreas (7 male and 3 female) wereused in this study. All pancreata were harvested, after obtainingappropriate consent, from cadaveric organ donors and transported in coldsolution of 30 mM raffinose, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mMMgSO₄, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, 8 mMdexamthazone and 5% (w/v) hydroxyethyl starch (HES), pH 7.4 (UWsolution). The age of donors ranged from 17 to 63 years (40.8±4.7 years)and cold ischemic time ranged from 3 to 11 hours (8.4±1.0 hours).

After intraductal injection of 150-250 ml of a solution of 5.6 mMglucose, 15 mM KH₂ PO₄, 0.33 mM Na₂ HPO₄, 0.82 mM MgSO₄, 5.4 mM KCl, 137mM NaCl and 1.3 mM CaCl₂, pH 7.2 (HBSS) containing 2.5 mg/ml collagenase(Collagenase P, lot #13494022-50, Boehringer Mannheim Colo.,Indianapolis Ind.) at 37° C., the pancreata was cut into approximately8-10 pieces (2-3 cm³) and placed in a disposable clear plastic container(500 ml in size), such as that shown in FIG. 1, with three glass marbles(1 cm in diameter). First, the pancreata was gently agitated with ashaker (Wrist Action Shaker Model 175, Burrell Co. Pittsburgh, Pa.) in a37° C. water bath for 15-20 minutes (Step 1).

The collagenase solution containing the digested tissue was filteredthrough a 8 mm screen and transferred to a second clear plasticcontainer to which 1/3 of the total amount of cold preservationsolution, 30 mM D-mannitol, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mMMgSO₄, 30,000 units/l superoxide dismutase, 5 mM nicotinamide, pH 7.3(LAP-1) was added. This container was then hand-shaken on ice to furtherdigest the tissues. Every 5 minutes, shaking was stopped, 1/3 of thesolution containing digested tissues was removed though a 800 μm screen,the same volume of fresh cold LAP-1 solution was added to the containerand the digestion continued (Step 2).

This process was repeated several times until most of the islets werefreed from the tissue. The undigested tissue that remained on the 8 mmscreen was further digested in a 37° C. water bath for an additional5-10 minutes until most of the remaining tissue consisted of pancreaticducts. Usually, this second warm digestion was sufficient to release theremaining acinar and endocrine components from the larger ductalstructures. This digested tissue was also transferred into the colddigestion container, for further cold digestion. The entire procedurewas completed within 60 minutes.

The following four solutions were used to store digested pancreatictissue in the cold: HBSS, UW solution, modified UW solution whichconsisted of the same components as UW solution, but omitting HES, andLAP-1 solution. The components of these solutions are compared in TableI.

                                      TABLE I    __________________________________________________________________________    COMPONENTS    SOLUTION           HBSS      mUW      UW       LAP-1    __________________________________________________________________________    Impermiants           5.6 mM glucose                     30 mM raffinose                              30 mM raffinose                                       30 mM D-                                       mannitol                     100 mM K-                              100 mM K-                                       100 mM K-                     lactobionate                              lactobionate                                       lactobionate    H.sup.+  buffers           15 mM KH.sub.2 PO.sub.4                     15 mM KH.sub.2 PO.sub.4                              15 mM KH.sub.2 PO.sub.4                                       15 mM KH.sub.2 PO.sub.4           0.33 mM Na.sub.2 HPO.sub.4    Metabolites           0.82 mM MgSO.sub.4                     5 mM MgSO.sub.4                              5 mM MgSO.sub.4                                       5 mM MgSO.sub.4    and others           5.4 mM KCl         5 mM adenosine                                       30,000 U/l SOD           137 mM NaCl        3 mM     5 mM                              glutathione                                       nicotinamide           1.3 mM CaCl.sub.2  1 mM                              allopurinol                     8 mM     8 mM                     dexamthazone                              dexzmthazone                              HES (5% w/v)    Na.sup.+  (mEq).sup.1           138       30       30       30    K.sup.+  (mEq)           5.8       120      120      120    mOsm/l.sup.2           284       320      320      320    pH     7.2       7.4      7.4      7.3    __________________________________________________________________________     .sup.1 mEq = milliequivalents     .sup.2 mOsm/l = milliosmolar/l

LAP-1 (100 mM K-lactobionate, 30 mM D-mannitol, 15 mM KH₂ PO₄, 5 mMMgSO₄, 30,000 U/l superoxide dismutase-SOD from bovine erythrocytes,Sigma Chemical Co., St. Louis, Mo. and 5 mM nicotinamide-Sigma ChemicalCo., St. Louis, Mo.) was adjusted to a pH of 7.3 at room temperature.Newborn calf serum was added to each solution at 20% (v/v) concentrationto store digested tissues.

Four separate 50 ml plastic tubes (Fischer Scientific, Pittsburgh, Pa.),each containing 25 ml of either HESS, UW, mUW, or LAP-1 were placed onice. Each time tissue was removed from the container during the colddigestion process, the tissue was dispensed equally between the fourtubes until an equal amount of digested tissue (a total of 1.5 to 2 ml)was placed in each tube. The tubes were then stored for 90 minutes onice. The remaining digested tissues were processed by a bulk isletisolation procedure, as described below.

After 90 minutes preservation at 4° C., digested tissue from each tubewas separately suspended in Euro-Ficoll solution (density of 1.100g/cm³) and purified by a discontinuous gradient centrifugation onEuro-Ficoll solutions (d=1.100, 1.087, 1.056) in HBSS at 450×g for 20minutes at 4° C. After centrifugation, cells were taken from eachdensity interface to identify the islet layer. Cells in the secondlayer, the interface between 1.087 and 1.056 and the third layer, theinterface between 1.100 and 1.087, contained islets. The islets, fromthe interfaces were collected and washed twice with the correspondingcold solution at 4° C., followed by one wash with RPMI1640 culturemedium (see below) at room temperature. Yield and purity of isletpreparations were evaluated by dithizone (DTH) staining. The isletnumber was expressed as 150 μm islet equivalents (IEQ).

Islets resuspended in RPMI1640 culture medium were distributed equallyinto six plastic Petri dishes (Falcon #1008, Becton Dickinson Co.,Franklin Lakes, N.J.) per group to be used for counting the isletnumbers on days 0, 1 and 3 following preparation. Additionally, threedishes of each group were prepared for evaluation of islet viability andfor assessment of glucose stimulated insulin release in a staticincubation assay and a perfusion system and the measurement of isletinsulin content. The medium used for culture was RPMI1640 supplementedwith 10% (v/v) fetal bovine serum (FBS), 10 mM nicotinamide, 25 mMHEPES, 24 mM NaHCO₃, 100 units/ml penicillin G, 100 μg/ml streptomycinand 0.25 μg/ml amphotericin B. All culture dishes were placed in atissue culture incubator in a 5% (v/v) CO₂ /air environment, at 37° C.

Islet Yield

Immediately after isolation, the total islet yield in each group wasevaluated, in duplicate, using DTH staining. The islet number wasconverted to 150 μm islet equivalents (IEQ). The result of each groupwas expressed as a percentage of the islet number yielded with HBSS ineach series.

Islet Purity After Isolation

The sample used for counting islet yield was also used to evaluate thepurity of islet preparations. The purity was estimated from theproportion of DTH stained and unstained cells. The result was expressedas a percentage of the DTH stained cell number to the total cell number.

Islet Numbers During Culture

Islet numbers in two randomly selected dishes were counted on days 0, 1and 3 (day 0 is the day of islet isolation) using DTH staining.

Viability of the Islets

On days 0, 1 and 3 of culture, the viability of islets was assessedusing supravital staining with fluorescein diacetate and ethidiumbromide as described by Gray et al., Stain Technology 62 373-381 (1987).The viability of each islet was scored as 0 (0%: dead islet), 1 (25%viable islets), 2 (50% viable islets), 3 (75% viable islets) and 4(100%: fully viable islet) and the percent viability was calculated fromthe following formula:

% viability=(0.25×number of islets scored as 1+0.5×number of isletsscored as 2+0.75×number of islets scored as 3+number of islets scored as4) divided by total islet number×100.

Insulin Release Assay

To assess β-cell function, static incubation tests were performed oneach group between day 3 and day 8. One thousand islets (IEQ) wereincubated in 1 ml RPMI1640 containing 60 mg/dl glucose (basal medium)for 45 minutes and then in 1 ml RPMI1640 containing 300 mg/dl glucose(high glucose stimulation medium) for 45 minutes. The islets were againincubated in 1 ml basal medium for 45 minutes. Insulin levels releasedinto each medium were measured by solid-phase radioimmunoassay (Autopakinsulin kit, ICN Biomedicals Inc., Costa Mesa, Calif.). A stimulationindex was calculated as the insulin released into the stimulation mediumdivided by insulin released into the basal medium. After the staticincubation test, insulin was extracted from the islets by overnightincubation in acid alcohol to measure islet insulin content.

In addition, dynamic insulin release in response to glucose stimulationwas evaluated by a perfusion system with the islets isolated using LAP-1cold preservation solution. Five hundred islets, placed in a cytodex gel(Cytodex 2, Pharmacia Inc, Piscataway, N.J.) column, were first perfusedat 37° C. with Krebs buffer solution containing 60 mg/dl glucose and 2%(v/v) heat-inactivated newborn bovine serum for 100 minutes to stabilizethe baseline. During the next 15 minutes, samples were collected everyone minutes (basal insulin release). The medium was then changed toKrebs buffer containing 300 mg/dl glucose and perfused for 30 minutesbefore changing to the basal buffer to observe prompt shut-off ofinsulin release. Insulin levels in each sample collected during theperfusion (1 minute/tube) were measured by solid phase radioimmunoassay.

Electron Microscopy of Islets

Immediately after isolation, islets were fixed with 1.25% (w/v)glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at room temperaturefor the ultrathin sectioning method. Islets were washed in 0.1 Mphosphate buffer containing 5% (w/v) sucrose (pH 7.4, 360 mOsm) andsuspended in 70% (v/v) calf serum in 0.1 M phosphate buffer, pH 7.4.Subsequently, cells were centrifuged at 450 g for 15 minutes to form apellet which was immersed in 2% (w/v) glutaraldehyde in 0.1 M phosphatebuffer (pH 7.4, 440 mOsm) for 2 hours. Each pellet was cut into smallblocks and washed with 0.1 M phosphate buffer containing 8% (w/v)sucrose (pH 7.4, 440 mOsm) followed by post-fixation in 1% (w/v) osmiumtetroxide in 0.1 M phosphate buffer containing 7% (w/v) sucrose (pH 7.4,440 mOsm) for 2 hours. The fixed specimens were dehydrated in a gradedseries of ethanol and embedded in epoxy resin. The thin sections werestained with uranyl acetate and lead citrate. Observations wereperformed with a Hitachi H-7000 transmission electron microscope.

Data Analyses

All data were expressed as mean±standard error unless otherwisespecified. Differences between groups were compared using an unpaired,two-tailed Student's t-test and were considered statisticallysignificant if p value <0.05.

Results

Immediately after islet isolation, groups mUW, UW and LAP-1 yieldedhigher numbers of islets than the HBSS group. The difference wasstatistically significant in groups mUW and LAP-1. There was a widerange of islet yields from one set of experiments to another, dependingon the volume of digested tissue used. For this reason, islet yieldswere expressed as percent of the value obtained with HBSS. Islet yieldsin group LAP-1, in particular, were twice as high as those in the HBSSgroup (192±92%), see Table II.

                                      TABLE II    __________________________________________________________________________    Raw data expressed as Islet                              Data expresses as a percentage    Equivalents (IEQ)         of the HBSS values    Sample          HBSS mUW  UW   LAP  HBSS mUW  UW   LAP    __________________________________________________________________________    Hu148  2675                8267                     2492                          6750                              100  309.05                                         93.17                                             262.34    Hu153 14775               17883                    16168                         18467                              100  121.04                                        109.36                                             124.99    Hu154 17608               31641                    18667                         19825                              100  180.72                                        106.62                                             113.23    Hu166 31592               22017                    46625                         40160                              100   69.69                                        147.68                                             127.09    Hu167 41808               69475                    38450                         46367                              100  142.26                                         91.97                                             110.9    Hu169 13442               19292                    11392                         22625                              100  143.62                                         84.75                                             168.32    Hu164  4050                8533                    13167                         14000                              100  210.69                                        325.11                                             346.68    Hu165  4930                8160                     7310                         12910                              100  165.62                                        148.28                                             261.87    Hu167-1          13687               22313                    25533                         42900                              100  163.02                                        186.66                                             313.44    Hu167-2          19380               21573                    23307                         20673                              100  111.32                                        120.26                                             106.67    Mean  16385               21916                    20310                         24467                              100  161.68                                        141.36                                             192.46    SD.sup.1          12372               15214                    13723                         13734                               0    64.87                                         71.91                                              92.02    __________________________________________________________________________     .sup.1 SD = standard deviation

¹ SD=standard deviation

The purity of islet preparations in the HBSS group was 41.5±14.8%. Incontrast, the purity in groups mUW, UW and LAP-1 was 71.5±8.9%,74.9±10.7%, 75.8±7.4%, respectively, which were significantly higherthan that of the HBSS group (p<0.001). Differences between these threegroups, however, were not significant. Highly purified islets (as highas 95% pure) were isolated with LAP-1. In contrast, islets in the HBSSgroup were contaminated with exocrine tissue and the purity was as lowas 25%.

In the HBSS group, the islet numbers decreased to 55.6±14.4% on day 1and 27.4±16.5% on day 3 as compared to that on day 0 (100%). Incontrast, significantly higher numbers of islets were counted in allother groups on both days 1 and 3 as compared to those in the HBSSgroup. Especially in group LAP-1, the islet number was maintained at thehighest level of 94.6±20.4% on day 1 and 78.5±24.2% on day 3, but asignificant difference was not obtained between groups mUW and UW.

At the time of islet isolation, the percent viability estimated bystaining with fluorescein diacetate and ethidium bromide was alreadylowest in the HBSS group (HBSS vs UW, HBSS vs LAP-1; p<0.05). Isletviability in this group continuously decreased to 37.4±19.5% on day 1and 16.8±13.2% on day 3, whereas the other three groups maintainedsignificantly higher islet viability. As shown by a electromicrograph ofa representative case, islet damage was clearly less in the LAP-1 groupthan that in HBSS.

Contamination with cell debris and acinar cells was more prominent inthe HBSS group as compared to LAP-1. In addition, islet cells in theHBSS group displayed a decreased density of cytoplasm, mitochondrialswelling and unclear or ruptured cell membranes. By contrast, thesefindings were consistently less frequent in the LAP-1 group.

The highest insulin content was found in islets isolated with LAP-1.However, no statistically significant differences were detected betweenany of the groups.

In groups mUW, UW and LAP-1, the stimulation indices were higher thanthat of the HBSS group. In particular, in groups mUW and LAP, theindices were significantly higher than that of the HBSS group.

Islets isolated in LAP-1 released insulin immediately after glucosestimulation with a two phase insulin release, which promptly ceased whenthe high glucose buffer was replaced by the basal buffer. The curvedemonstrated the normal function of β-cells.

The two-step digestion technique for the human pancreata has been usedsuccessfully to isolate a large number of viable islets. The techniqueinvolves two phases of digestion processes, first at 37° C. and then onice. In cold digestion, the collagenase solution was gradually replacedby cold preservation solution. The tissue is broken down into smallerfragments by warm collagenase digestion, it continues to digest on iceinto fine fragments and finally to free islets. Islets prepared by thetwo-step procedure maintained a well preserved capsule and an excellentthree-dimensional structure. This method prevents overexposure of isletsto collagenase and thus avoids over-digestion of islets as well asacinar cells.

UW solution is the most advanced and widely used cold preservationsolution for storing organs awaiting transplantation. UW solutioncontains a low concentration of Na⁺ and high concentration of K⁺,similar to the composition of intracellular fluid. In addition, UWsolution contains the lactobionate anion and raffinose as impermeablecomponents and HES to maintain the colloid pressure. This compositionprevents cell swelling that occurs when cells are stored in conventionalphysiological solutions (i.e. HBSS) in the cold, a condition in whichthe cell membrane sodium-potassium pump ceases to function. Although HESis an important component for maintaining vascular networks when anorgan is flushed out with cold preservation solution, it may be omitted,with no deleterious effect on cell viability, in cold storage of smallorgans or tissues. We therefore modified the UW solution to contain onlylactobionate-K⁺, raffinose, KH₂ PO₄ and MgSO₄ and compared theeffectiveness of both mUW and UW in this study. Along with these tests,we also developed a new cold preservation solution, LAP-1, for isletswith special attention to their vulnerability. LAP-1 solution containslactobionate and D-mannitol as impermiants, to inhibit cell swelling bymaintaining extracellular osmotic pressure. D-mannitol is also known tohave a scavenging activity for hydroxy radicals (OH·) to which β-cellsare highly susceptible. As with UW solution, LAP-1 solution contains KH₂PO₄ as a component of the buffering system. In addition, the solutioncontains SOD (30,000 units/l) and nicotinamide (5 mM/l). Our expectationis that SOD and nicotinamide would enter islet cells, that may have beendamaged during the isolation process, to protect them from furtherdamage and to support their recovery in culture or after beingtransplanted.

In this study, the performance of four solutions for storing digestedpancreatic tissue at low temperature have been compared by evaluatingthe yield, the purity of the recovered islets and the viability ofislets maintained in culture. Cell viability was also assessed byelectron microscopic examination of islets and by β-cell function. Theyield and purity of isolated islets were markedly improved by usingsolutions especially designed for cold storage of organs (UW, mUW,LAP-1), instead of using cold HBSS. Although HBSS has been widely usedfor human islet isolation, the first interface between HBSS and 1.056g/cm³ Euro-Ficoll contained many fragmented islets and the secondinterface between 1.056 and 1.088 g/cm³ Euro-Ficoll contained mostislets and a considerable number of exocrine cells. This indicated thatthe density of both islet and acinar cells had changed significantly,probably due to cell swelling during cold storage. This resulted in moreislet destruction (thus, lower yield) and lower islet purity. Incontrast, with other solutions, almost all islets were found in thesecond interface with far fewer acinar cells, indicating that UW, mUWand LAP-1 solutions prevented cell swelling and had maintained thedensity of both islet and exocrine tissues. Electron microscopicexamination of islets isolated with LAP-1 solution revealed no cellswelling and a normal structure of islet cells. Since the islet yieldand purity were not significantly different between these threesolutions, HES and raffinose may not be essential components of the coldpreservation solution for preventing cell swelling under the conditionsused in the islet isolation.

Islet numbers were also counted during culture for three days. The isletnumber on day 1 was especially important, since it probably reflects anactual islet number that may survive following transplantation. On day1, the islet numbers in the HBSS group had already decreased by nearly50% and declined to only 25% of the initial count on day 3. This declineindicated severe and often irreversible damage to islets that occurredduring the cold storage of pancreatic digests. Islet numbers in theother three groups were significantly higher at all points, rangingbetween 95% and 75% on day 1 and between 78% and 60% on day 3, ascompared to those on day 0. The decreased islet numbers and viability onday 3 in this study may have been influenced by the culture conditions,since only a small volume of culture medium was used in order toaccommodate islet counting. Although no significant differences werefound between these three groups, islet viability was higher in groupsUW and LAP-1. These results show that the prevention of cell swellingduring the islet isolation process contributes not only to theimprovement of islet yield and purity, but also to greater isletviability. The results of β-cell functional tests correlated with theseviability results. The stimulation index was highest in the LAP-1 group.However, islet insulin content, as expressed by μU/islet (IEQ), did notshow any differences between the groups. This is not surprising, sincean equal number of islets, that survived for more than 3 days inculture, was used for these tests. This also indicates that isletsrecover fully in culture by day 3 and survive.

The beneficial effects of UW, mUW and LAP-1 solutions over HBSS havebeen clearly demonstrated in the preparation of human pancreatic islets.Among these, LAP-1 solution was most effective, providing the bestyield, purity and viability of isolated islets. Also, the high viscosityof the UW solution made it undesirable for use in the isolation ofislets as manipulations with the viscous solution were difficult. In aperfusion system to test dynamic insulin release in response to highglucose stimulation, islets in the LAP-1 group exhibited excellentfunctional responsiveness.

Pancreatic islets have two different characteristics: one as a cell andanother as an organ. The islet acts as a miniature organ, since itconsists of several different cell types which interact functionallywith one another and has its own vascular system. In order to maintainnormal islet function, islet structure needs to be preserved carefully.The collagenase digestion process is a procurement process of isletsfrom the larger organ, the pancreata, under warm ischemic conditions.The subsequent processes, that include cold storage, purification andwashing of islets, correspond to the cold anaerobic preservation of adonor organ. Based on the well documented theory for organ harvesting,shortening of the warm ischemic process is the first critical issue andthe use of an appropriate preservation solution for cold storage is thesecond critical issue in the procurement of highly viable donor organs.The intermittent two-step digestion procedure to minimize warm anaerobicischemia and the use of LAP-1 solution for cold preservation of digestedpancreatic tissue, both follow this basic theory. Their use resulted inhigher yield, purity and viability of islets from the human pancreata.

Human islets were isolated by an intermittent two-step collagenasedigestion procedure with warm collagenase digestion of the pancreata inless than 20 minutes. The cold digestion process and the cold storage ofdigested tissue (containing islets) should use an appropriate coldpreservation solution. LAP-1 was designed as a cold preservationsolution especially for islets; it showed clear advantages in thepreparation of high quality islets and is also economical for use in alarge quantity.

EXAMPLE 2 Preparation of Donor Pancreas

A total of 46 human pancreata were processed consecutively for isletsusing the new isolation procedures, described above in Example 1 (group1). Islet preparations from 46 pancreata processed by a conventionalisolation procedure served as the control group (group 2).

All pancreata were harvested with appropriate consent from cadavericorgan donors through the two organ procurement agencies of SouthernCalifornia. Procurement was performed by the standard techniques, byeither the members of the UCLA Liver Transplant Program or surgeons fromother institutions. All organs used in this study were a part of thepancreas containing the body and tail. The mean donor age of group 1 was33±3.2 years and ranged from 5 to 64 years; in group 2, the mean age was36±2.7 years, ranging from 11 to 69 years and there were no significantdifferences between two groups. The pancreata was transported to thelaboratory in either UW solution or Euro-Collins solution. Cold ischemictime in groups 1 and 2 was 8.1±0.4 hours and 6.6±0.5 hours,respectively, and not significantly different. The organ was trimmed ofsurrounding membranes, vessels, fat and lymph nodes, cannulated with anangiocatheter and weighed. The organ was expanded by injecting 150-250ml of warm collagenase solution using a 30 ml syringe and sectioned intoapproximately 8-10 pieces. The collagenase solution containedCollagenase P (Boehringer Mannheim Colo.., Indianapolis, Ind.), 2% (v/v)heat-inactivated newborn bovine serum (NBS, Sigma Chemical Co., StLouis, Mo.), 1 mg/ml calcium chloride and DNase in HBSS. Theconcentration of collagenase was optimized for each lot and ranged from1.5 to 2.5 mg/ml.

For group 2, pancreatic tissues were enzymatically digested by aone-step procedure in a 37° C. water bath. The procedure has beendescribed in detail by Benhamou et al., Transplantation 57 1804-1810(1994). Briefly, the sectioned pancreas was placed in a 5×7 cmstainless-steel, enclosed mesh container containing three 1.5 cm glassmarbles. The container was then placed into a disposable clear plasticchamber and the tissue was covered by collagenase solution. The totalcollagenase volume, used both for expansion of the organ and solutionplaced in the container, was approximately 200 ml. The chamber wasagitated in a 37° C. water bath via a swing-arm shaker (Wrist ActionShaker, Model 75, Burrell Co., Pittsburgh, Pa.). Samples were taken atregular intervals. When free islets were noted, usually within 15minutes after the start of digestion, the collagenase and digestedtissue mixture were removed from the chamber. Sufficient fresh warmcollagenase solution was added to the chamber to cover the remainingtissues and the digestion was continued. This process was repeated every5 minutes until no more free islets were detectable, or until alltissues were digested. The total process required 45 to 60 minutes andthe total collagenase solution was up to 600 ml. Thecollagenase/digested tissue mixture decanted from the container wasimmediately diluted with cold HBSS containing 20% (v/v) NBS and storedon ice until digestion was completed.

For group 1, tissues were digested by a two-step procedure, first in a37° C. water bath, then followed by cold digestion of the partiallydigested tissues on ice. Pieces of the pancreas were placed in adisposable, clear plastic container containing three glass marbles. Thechamber was gently agitated in a 37° C. water bath via a swing-armshaker as above, for 15 minutes, until the pancreas was partiallydigested (Step 1: warm digestion phase). Collagenase solution containingpartially digested tissues was filtered through a sheet of large-mesh (8mm) screen into a second clear plastic container placed on ice. ColdLAP-1 preservation solution was added to the second container at avolume approximately 1/3 of the decanted volume. In the first container,warm collagenase was again added to cover the remaining tissue and warmdigestion was repeated. After 5 minutes, all collagenase/digestedtissues in the first container were transferred into the secondcontainer. The second container was connected to a collection bottle andthe cold preservation solution via two separate side ports. The secondcontainer was gently hand-shaken on ice. Every 5 minutes, 1/3 of thesupernatant containing digested tissues was decanted, through anout-flow port, into a collection bottle containing cold LAP-1 solutionsupplemented with 20% (v/v) NBS. LAP-1 solution was added to the secondcontainer, via another port, at a volume equal to that decanted and thecontainer was shaken again. This cold digestion process was repeateduntil all islets were freed (Step 2: cold digestion phase). Ifnecessary, warm digestion was repeated a third time, so that onlyundigested, larger ductal structures remained.

To avoid harmful effects on digested tissues, collagenase and otherenzymes released from pancreatic acinar cells must be inactivated assoon as possible. For this purpose, finely digested pancreatic tissuescontaining free islets were collected at 5-10 minute intervals andstored on ice in a large amount of cold storage solution. In group 1,pancreatic digests were stored in cold LAP-1 solution supplemented with20% (v/v) NBS, whereas in group 2, tissues were stored in cold HBSScontaining 20% (v/v) NBS.

Pancreatic digests in both groups were suspended in Euro-Ficoll solutionwith a density of 1.100 g/cm³ and islets were separated from acinarcells by discontinuous gradient centrifugation on three layers ofEuro-Ficoll solutions (densities of: 1.100, 1.087 and 1.056) using aCOBE2991 cell processor (Cobe Laboratories, Inc., Lakewood, Colo.).After 10 minutes centrifugation, islet fractions were identified andcollected. Islets were washed twice with cold LAP-1 solution and oncewith RPMI1640 medium containing 10% (v/v) fetal bovine serum (FBS) atroom temperature.

Isolated islets were cultured in plastic Petri dishes (Falcon #1005,Becton Dickinson Co., Franklin Lakes, N.J.) at 37° C. with RPMI1640containing 20% (v/v) FBS, 10 mM nicotinamide, 25 mM HEPES, 24 mM NaHCO₃,100 units/ml penicillin G, 100 mg/ml streptomycin and 0.25 mg/mlamphotericin B.

Temperature

The temperature of the digestive medium during the cold digestion phasein the intermittent two step digestion process (group 1) was monitoredevery 5 minutes in five isolation cases.

Islet Yield

Immediately after isolation, the total islet yield was evaluated induplicate by taking a known volume of sample and staining with dithizone(DTH). The islet number was counted, each size separately and the totalislet number converted to that equivalent to islets 150 μm in size(IEQ). The islet yield was expressed as the total islet number isolatedfrom a given pancreas and the number of islets per gram of pancreatictissue.

Islet Purity

The purity of the islet preparation was evaluated on the same sample asthat used for islet counting and was calculated as the ratio betweenDTH-stained and total cells, expressed as a percentage.

Assessment of Viable Islets After 48 Hours in Culture

Islets that remained viable in culture for 2 days were taken as "viable"islets that would survive, in vivo, after transplantation. This wasevaluated by placing islets in culture and assessing their viability 48hours after isolation. Islet viability was determined using DTH forcounting and fluorescein diacetate and ethidium bromide (FDEB) stainingfor viability assessment as described by Gray et al., Stain Technology62 373-381 (1987).

In Vitro Insulin Release Assays

To assess the β-cell function of cultured islets, two glucosestimulation tests, static incubation and a dynamic insulin release assayin a perfusion system, were performed on several randomly selectedpreparations in group 1.

i) Static incubation assay:

One thousand islets (IEQ) were incubated successively in basal,stimulation and basal medium as described above. RPMI1640 medium wasused with the glucose level reduced to 60 mg/dl for the basal medium andincreased to 300 mg/dl for the stimulation medium. Insulin releasedduring each incubation was measured by a solid-phase radioimmunoassayusing human insulin as standard (Autopak Insulin Kit, ICN BiomedicalsInc., Costa Mesa, Calif.).

The stimulation index was calculated as: insulin released into thestimulation medium divided by insulin released into the basal medium.ii) Dynamic insulin release assay in a perfusion system: Five hundredislets (IEQ) were layered in a cytodex bead column, which was placed ina 37° C. water bath and perfused successively in the following order:basal, stimulation and basal medium as described previously by Benhamouet al. (Hormone & Metab. Res. 27 113-120, 1995). Media used in theperfusion system were Krebs Ringer bicarbonate buffer solutionscontaining 60 mg/dl (basal) and 300 mg/dl (stimulation) glucose,respectively. Insulin was assayed as for static incubation.

In Vivo Islet Function Test

The ability of islets to reverse diabetes was assessed, in vivo, bytransplanting them into athymic mice made diabetic with an intravenousinjection of 165 mg/kg streptozotocin. Islets (1,000-1,200 IEQ) fromgroup 1, cultured for 3 days, were transplanted under the left renalcapsule of diabetic mice. After transplantation, levels of non-fastingblood glucose, urine glucose and body weight were monitored three timesa week. Glucose tolerance tests were also performed on day 14 byinjecting 0.5 g/kg glucose into the tail vein and a K-value wascalculated using blood glucose levels at 5, 10, 30 and 60 minutes afterglucose challenge. On day 20 or 21, mice underwent a left nephrectomy toconfirm the recurrence of diabetes and grafts were examinedhistologically.

Statistical Studies

All data were expressed as mean value±standard error of the mean and thedifference between groups 1 and 2 was considered significant if thep-value was less than 0.05, using a two-tailed unpaired student t-test.The significance of differences in viable islet recovery was examinedusing a chi-square test.

There were no significant differences between groups 1 and 2 with regardto the donor age, the cold ischemia time and harvesting conditions. Thepancreas weight was also similar in both groups; 55±2.7 g in group 1 ascompared to 56±3.4 in group 2.

                  TABLE III    ______________________________________           Pancreas                   Digestion Time    Group  n.sup.1                 Weight (g)                           Warm    Cold    Total    ______________________________________    1      46    55 ± 2.7                           21.5 ± 0.7                                   41.7 ± 1.2                                           59.3 ± 2.5    2      46    56 ± 3.4                           56.1 ± 2.2                                   --      56.1 ± 2.3    p-value      N.S.sup.2 p < 0.001                                   --      N.S.    ______________________________________     .sup.1 n = number of isolations     .sup.2 N.S. = Not significant

As shown in Table III, the duration of warm digestion was significantlyless in group 1 (21.5±0.7 minute) compared to that in group 2 (56.1±2.3minute). In group 1, warm digestion was followed by cold digestion for41.7±1.2 minute, so that the total time required to digest the pancreaswas 59.3±2.5 minute, similar to that of group 2.

Temperature of the medium was measured during the cold digestionprocedure (n=4) Table IV.

                  TABLE IV    ______________________________________    Time    Average        Time    Average    (min.)  Temp. (°C.)                           (min.)  Temp. (°C.)    ______________________________________     0.sup.1            37             30      15     5      37             35      12    10      37             40      10    15      37             45      9     20.sup.2            30             50      7    25      20             55      6    ______________________________________     .sup.1 Time 0-15 min. : Warm digestion process     .sup.2 Time 20-55 min. : Cold digestion process

Also, the change in the collagenase concentration during this processwas calculated based on the amount of LAP-1 solution added each time,see Table V.

                  TABLE V    ______________________________________    Time    Collagenase    Time    Collagenase    (min.)  (mg/ml)        (min.)  (mg/ml)    ______________________________________     0.sup.1            2.5            30      0.75     5      2.5            35      0.50    10      2.5            40      0.30    15      2.5            45      0.20     20.sup.2            1.6            50      0.15    25      1.2            55      0.10    ______________________________________     .sup.1 Time 0-15 min. : Warm digestion process     .sup.2 Time 20-55 min. : Cold digestion process

By the repeated replacement of warm collagenase solution with cold LAP-1solution during the cold digestion process, the temperature of thesolution rapidly declined from the initial 37° C. to 15° C. in less than30 minutes, then to <10° C. by 40 minutes. By dilution calculations, thecollagenase concentration also decreased from 2.5 mg/ml to 0.1 mg/ml bythe end of the digestion period.

Islets were successfully isolated from 44 of the 46 pancreata in group 1(success rate: 95.6%) and from 33 of the 46 pancreata in group 2 (71.7%;p<0.01). In 9 of the 13 failures in group 2, the isolation process wasterminated before the islet purification step with the COBE2991 cellprocessor due to an insufficient number of islets and their poorappearance during digestion. In the remaining 4 cases, most of theislets disappeared or disintegrated after the purification process.

In group 1, the total islet yield was also significantly higher thanthat of group 2 as shown in Table VI.

                  TABLE VI    ______________________________________    Total Yield.sup.1 Yield/g Pancreas.sup.1    Group    Cells × 10.sup.3                          Group    Cells × 10.sup.3    ______________________________________    1 (n.sup.2 = 44)             336 ± 36  1 (n = 44)                                   6.2 ± 0.7    2 (n = 33)             196 ± 25  2 (n = 33)                                   3.6 ± 0.3    ______________________________________     .sup.1 Islet numbers shown by IEQ     .sup.2 n = number of isolations

The islet yield in group 1 was 335,739±36,244 IEQ from the body and tailof the pancreas (pancreas weight: 33±3.2 g) and 6,233±681 IEQ/g oftissue, while that in group 2 was 195,587±25,242 IEQ per pancreas(pancreas weight: 36±2.7 g) and 3,763±509 IEQ/g (p<0.01). The purity ofisolated islets was 83.6±2.5% in group 1 and 69.2±4.7% in group 2(p<0.05), see Table VII.

                  TABLE VII    ______________________________________           Group  Purity    ______________________________________           1 (n.sup.1 = 44)                  84 ± 3           2 (n = 33)                  69 ± 5    ______________________________________     .sup.1 n = number of isolations

Islets placed in culture were viable, as assessed by FDEB staining,after 48 hours in 43 of the 44 successful isolations in group 1, seeTable VIII.

                  TABLE VIII    ______________________________________    Group          Viable    Non-Viable    ______________________________________    1 (n.sup.1 = 44)                   43 (97.7%)                             1 (2.3%)    2 (n = 33)     26 (78.8%)                             7 (21.8%)    ______________________________________     .sup.1 n = number of isolations

Thus, overall, 43 of a total of 46 pancreata prepared by our newprocedure provided viable islets as assessed by highly strict criteria.With these criteria, the success rate in group 1 was 93.5%. In contrast,islets in group 2 were viable after 2 days in culture in 26 of the 33successful isolations (see Table VIII). Thus, the overall success ratewas 56.5% (26 of the 46) in this series of isolations. The differencebetween groups 1 and 2 was significant (p<0.001).

Static incubation assays were performed on six different isletpreparations in group 1. Stimulation indices calculated from theseexperiments are shown in Table IX.

                  TABLE IX    ______________________________________                  Stimulation    Exp. No.      Index    ______________________________________    Hu153         1.91    Hu154         1.91    Hu164         3.03    Hu166         1.09    Hu167         2.65    Hu168         1.15    Mean ± SE  1.96 ± 0.32    ______________________________________

Islets in all preparations responded to high glucose stimulation,although the stimulation indices were lower in #166 and #168. Asrepresented by preparation #196 (see Table X), dynamic perfusion studiesalso demonstrated normal β-cell function of islets prepared in group 1as shown by immediate and two phase insulin release after a high glucosechallenge and the prompt increase in insulin release when the medium waschanged to basal.

                  TABLE X    ______________________________________    T.sup.1        G.sup.2                          IRI.sup.3    ______________________________________    2              60     48    4              60     50    6              60     40    8              60     55    10             60     54    12             60     45    14             300    160    16             300    145    18             300    155    20             300    165    22             300    170    24             300    164    26             300    160    28             300    155    30             300    160    32             300    164    34             300    125    36             300    130    38             300    125    40             300    110    42             60     108    44             60     100    46             60     85    48             60     70    50             60     68    52             60     68    54             60     55    56             60     52    58             60     45    60             60     52    62             60     45    64             60     65    66             60     45    68             60     50    70             60     30    72             60     28    74             60     32    76             60     30    78             60     30    80             60     30    ______________________________________     .sup.1 T = Time (minutes)     .sup.2 G = Glucose (mg/dl)     .sup.3 IRI = Immunoreactive insulin (μU/ml)

Reversal of diabetes in athymic mice was achieved by transplantation of1,000-1,200 islets from group 1, see Table XI.

                  TABLE XI    ______________________________________    NFBG.sup.1                NFBG.sup.1    Day   Mouse 1   Mouse 2   Day   Mouse 1 Mouse 2    ______________________________________    -5    100       --         6    140     140    -3    --        110       10    --       60    .sup. 0.sup.2          360       350       15     60      95    1     --        195       21    .sup. 100.sup.3                                            --    2     195       --        22    280     .sup. 120.sup.3    3     --        120       23    --      400    4     120       --        24    390     --    ______________________________________     .sup.1 NFBG = nonfasting blood glucose     .sup.2 day of transplant     .sup.3 day of nephrectomy (graft removal)

Mice became normoglycemic (<150 mg/dl) with negative urine glucosewithin 5 days after grafting. K-values of the IVGTT were0.30±0.02%/minutes. Histological examination with hematoxylin-eosinstaining revealed well-formed islets and well-preserved β-cells asindicated by insulin staining. These in vivo results demonstratedsurvival and function of islets after transplantation.

Islet transplantation has not yet become a routine procedure fordiabetic kidney recipients, due to a low success rate and the relativelyshort period of insulin independence. Islet isolation from donorpancreas is the initial process crucial to realizing successful islettransplantation. At present, simultaneous kidney and islettransplantation for type I diabetic patients with end stage renalfailure would be the most reasonable form of islet transplantationbecause of the need for immunosuppression. In these patients, the use ofislets isolated solely from the pancreas of the kidney donor would bethe best approach to minimizing rejection by avoiding additionalantigenic disparities. To make this possible, a large number of isletsneed to be isolated from a single human pancreas. Another importantissue for successful islet transplantation is the ability to obtain highquality islets which survive and function well after grafting.

There are several factors that affect islet viability during isletpreparation. Donor factors, the surgical techniques for the removal ofthe pancreas and the preservation solution used for transporting theprocured pancreas can influence islet viability and recovery.Hospitalization period of the donor also affects the isolation outcome.In the islet isolation process, the most critical procedure is that ofthe collagenase digestion. This process is essential for separation ofislets from the surrounding tissues, but, at the same time, islets canbe damaged by both warm ischemic injury and enzymatic digestion mediatedby collagenase and possibly enzymes released from acinar cells. Anothercritical procedure is the handling of islets during the cold phase ofthe isolation process, which involves cold storage of pancreaticdigests, the islet purification process and cell washing. If notproperly processed, islets may suffer cell swelling and cold ischemicinjury. Additionally, islet survival can be affected by mechanicaldamage induced by pipetting and cell washing.

Collagenase distention of the pancreas and the digestion of tissues arean essential process for freeing islets, but are also harmful for bothislets and acinar cells. In order to minimize the toxic effect ofenzymes, the following three modifications are necessary: i) to reducewarm digestion time, ii) to minimize the exposure of freed islets towarm enzyme solution and iii) to stop enzyme activities by immediatelystoring digested tissues and freed islets in a suitable coldpreservation solution. In this study, we have demonstrated thatprolonged warm collagenase digestion is not necessary in order to freeislets. The exposure of pancreatic tissues to warm collagenase can bereduced to less than 25 minutes. Tissue digestion continues on ice andreleases islets. With the isolation technique of the present invention,the warm digestion time was significantly reduced from 50 minutes,required by earlier methods, to approximately 20 minutes. Since most ofthe islets were freed during the cold digestion process, theintermittent two-step procedure minimized the direct exposure of isletsto warm, concentrated enzyme solution. Moreover, during cold digestionthe enzyme solution was gradually replaced by the cold preservationsolution and thus, islets were released into cold, diluted enzymesolution, which was further diluted by the cold preservation solutionwhen digested tissues were stored.

The temperature of the digestion medium was rapidly lowered to 5-7° C.during the cold digestion phase. Also, the concentration of collagenasein the digestion medium decreased to 0.1 mg/ml based on the dilutioncalculations. The results indicate that free islets may disintegrate byenzyme action during the warm digestion process. It was also noted thatthe amount of collagenase used in the two-step digestion was less thanthat used in the one-step procedure (up to 300 ml vs. 500-600 ml). Bycombining the two-step digestion procedure and the use of LAP-1 solutionas a cold preservation solution, the success rate for obtaining viableislets markedly improved to 93.5% (43 of the 46 consecutive isolations)from 56.5% (26 of 46) prepared by our old method (as assessed after 48hours in culture). Prior to this study, these isolation failures wereattributed to various conditions including the donor's physicalcondition, medications, procurement techniques and/or prolonged coldischemia time. Although these factors influence the outcome of isletisolation, the results in the present study suggest that the failure ofisolation, poor viability of islets and lower islet yields can alsoresult from prolonged warm collagenase digestion and cold ischemicinjury during cold preservation in HBSS. It would also appear that thenew isolation technique can isolate islets from a damaged pancreas byproviding conditions that avoid further islet damage and preserve viableislets, whereas islet damage may be accelerated by the conventionaltechnique.

Both islet yield and purity were improved in group 1. After densitygradient centrifugation, islets were located in a confined area of theEuro-Ficoll, separate from the acinar cells. In contrast, islets andacinar cells in group 2 were distributed in a broader range ofEuro-Ficoll layers. The prevention of cell swelling also increases isletyields. All pancreata used in both groups 1 and 2 were partial,containing only the body and tail. On separate occasions, islets wereisolated from the whole pancreas using the newly developed technique. Inall cases greater than 500,000 IEQ were isolated. This number of isletsfulfills the requirement for transplantation currently recommended fortype I diabetic patients by providing >6,000 IEQ/kg of recipient bodyweight, >90% purity and >95% viability. The recovery of viable isletsafter 48 hours in culture is an important index for the prediction ofislet survival following transplantation. In group 2, 20% ofsuccessfully isolated islet preparations (as assessed immediately afterisolation) died after being placed in culture, suggesting thatirreversible islet damage occurred during the isolation process. Incontrast, islets in only one of the 44 successful isolations in group 1were lost in culture and islets were fully viable in the remaining 43cases. These islets responded with normal insulin release in both invitro and in vivo assays.

Although the two-step digestion procedure appears complex, the colddigestion process can be performed in a totally enclosed system, similarto that designed for our old method. The process is economical in regardto both time and personnel. The entire procedure can be carried out bytwo or three trained individuals within 4 to 5 hours, from the start ofcleaning of the pancreas to either placing isolated islets in culture orcompleting the packaging for transplantation. Thus, simultaneous isletand kidney transplantation is possible. With this technique, asufficient number of islets to fulfill the transplantation requirementcan be isolated from one pancreas and simultaneous kidney and islettransplantation is possible from a single donor.

All references cited above are hereby incorporated herein by referencein their entirety.

The above descriptions of exemplary embodiments of are for illustrativepurposes. Because of variations which will be apparent to those skilledin the art, the present invention is not intended to be limited to theparticular embodiments described above. The present invention may alsobe practiced in the absence of any element not specifically disclosed.The scope of the invention is defined by the following claims.

What is claimed is:
 1. A method of isolating islets from a pancreas, themethod comprising:at least partially digesting the pancreas in a warmcollagenase solution to form a warm digest; adding a cold preservativesolution to the warm digest to form a mixture; the cold preservativesolution comprising D-mannitol, at least 80 mM K-lactobionate, and abuffer; agitating the warm digest and preservative solution mixture at atemperature of about 0° C. to about 15° C. to cool and form a colddigest comprising solid and liquid material which comprises thepancreatic islets; and separating said islets from the cold digest. 2.The method of claim 1 wherein the step of at least partially digestingthe pancreas in the warm collagenase solution is carried out at atemperature of about 35° C. to about 38° C.
 3. The method of claim 1wherein the warm collagenase solution comprises Collagenase P, about 2%(v/v) heat-inactivated newborn bovine serum, about 1 mg/ml CaCl₂ and 0.4mg/ml DNase in a buffer selected from the group consisting of 5.6 mMglucose, 15 mM KH₂ PO₄, 0.33 mM Na₂ HPO₄, 0.82 mM MgSO₄, 5.4 mM KCl, 137mM NaCl and 1.3 mM CaCl₂, pH 7.2; 30 mM raffinose, 100 mMK-lactobionate, 15 mM KH₂ PO₄, 5 mM MgSO₄, 5 mM adenosine, 3 mMglutathione, 1 mM allopurinol and 8 mM dexamthazone, pH 7.4; 30 mMraffinose, 100 mM K-lactobionate, 15 mM KH₂ PO₄, 5 mM MgSO₄, 5 mMadenosine, 3 mM glutathione, 1 mM allopurinol, 8 mM dexamthazone and 5%(w/w) hydroxyethyl starch, pH 7.4; and 30 mM D-mannitol, 100 mMK-lactobionate, 15 mM KH₂ PO₄, 5 mM MgSO₄, 30,000 units/1 superoxidedismutase and 5 mM nicotinamide.
 4. The method of claim 1 wherein thecold preservative solution further comprises a membrane stabilizer, aradical scavenger, and nicotinamide.
 5. The method of claim 1 whereinthe cold preservative solution further comprises MgSO₄, superoxidedismutase, and nicotinamide.
 6. The method of claim 1 wherein the coldpreservative solution comprises about 25 mM to about 50 mM D-mannitol,about 80 mM to about 120 mM K-lactobionate, and about 15 mM KH₂ PO₄. 7.The method of claim 6 wherein the cold preservative solution furthercomprises about 1 mM to about 5 mM MgSO₄, up to about 30,000 units/litersuperoxide dismutase, and up to about 5 mM nicotinamide.
 8. The methodof claim 1 wherein the step of at least partially digesting the pancreascomprises:contacting the pancreas with a first portion of the warmcollagenase solution; digesting the pancreas in the first portion of thewarm collagenase solution to form a first portion of the warm digest andundigested pancreas; separating the undigested pancreas from the firstportion of the warm digest; contacting the undigested pancreas with asecond portion of the warm collagenase solution; digesting the pancreasin the second portion of the warm collagenase solution to form a secondportion of the warm digest and indigested pancreas; separating theundigested pancreas from the second portion of the warm digest;repeating the contacting, digesting, and separating steps until theundigested pancreas comprises undigested ductal structures; andcombining ail of the portions of the warm digest.
 9. The method of claim1 wherein said separating of the pancreatic islets from the cold digestcomprises:separating the solid material from the liquid material in thecold digest; wherein the liquid material comprises isolated islets inthe form of cell clumps less than 15 mm in size; adding a coldpreservative solution to the solid material; agitating the coldpreservative solution and solid material at a temperature of about 0° C.to about 15° C. to form a second cold digest comprising solid materialand liquid material; repeating the separating, adding, and agitatingsteps until the solid material comprises no pancreatic islets.
 10. Themethod of claim 1 wherein the step of adding said cold preservativesolution comprises adding the preservative solution at a temperature ofabout 0° C. to about 15° C. to the warm digest.